CSHL Press News

ChIP-Seq, Drosophila targeted mutagenesis featured in CSH Protocols

06/01/2009

COLD SPRING HARBOR, N.Y. (Monday, June 1, 2009) – High-throughput whole-genome analysis is becoming a standard laboratory approach for investigating cellular processes. Next-generation sequencing is replacing microarrays as the technique of choice for genome-scale analysis, because it offers advantages in both sensitivity and scale. The June issue of Cold Spring Harbor Protocols (www.cshprotocols.org/TOCs/toc6_09.dtl) features Native Chromatin Preparation and Illumina/Solexa Library Construction from Keji Zhao and colleagues at the National Heart, Lung and Blood Institute (http://public.nhlbi.nih.gov/Staff/Home/UserInputForPerson.aspx?OID=895&LabId=lmi). The article describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Solexa/Illumina Genome Analyzer. Step-by-step instructions are given for purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing. The article is freely available on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/6/pdb.prot5237).

Mutational analysis has long been a valuable tool for deciphering gene function. However, systematic repeated targeting of a single locus is difficult and is not a routine approach in multicellular organisms. Yikang Rong and colleagues at the National Cancer Institute (http://ccr.cancer.gov/Staff/Staff.asp?profileid=6230) have developed the Site-specific Integrase mediated Repeated Targeting (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. SIRT targets a landing site for the phage phiC31 integrase and allows the generation of several genetic variants at a locus of interest without having to perform multiple experiments. SIRT requires the construction of a series of plasmid vectors with varying arrangements of DNA elements. By taking advantage of bacterial recombineering approaches, SIRT bypasses the shortcomings of traditional cloning techniques that rely on the availability of convenient restriction enzyme cut sites. This method, SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila, is freely accessible on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/6/pdb.prot5236).

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About Cold Spring Harbor Protocols:Cold Spring Harbor Protocols (www.cshprotocols.org) is a monthly peer-reviewed journal of methods used in a wide range of biology laboratories. It is structured to be highly interactive, with each protocol cross-linked to related methods, descriptive information panels, and illustrative material to maximize the total information available to investigators. Each protocol is clearly presented and designed for easy use at the bench—complete with reagents, equipment, and recipe lists. Life science researchers can access the entire collection via institutional site licenses, and can add their suggestions and comments to further refine the techniques.

About Cold Spring Harbor Laboratory Press: Cold Spring Harbor Laboratory Press is an internationally renowned publisher of books, journals, and electronic media, located on Long Island, New York. Since 1933, it has furthered the advance and spread of scientific knowledge in all areas of genetics and molecular biology, including cancer biology, plant science, bioinformatics, and neurobiology. It is a division of Cold Spring Harbor Laboratory, an innovator in life science research and the education of scientists, students, and the public. For more information, visit www.cshlpress.com.